In vitro Effect of Photodynamic Therapy with Curcumin and Methylene Blue Photosensitizers on Staphylococcus Aureus

Statement of the Problem: Staphylococcus aureus (S.A) can colonize in the skin, nasal cavity, and oral cavity. In the oral cavity, it can cause dental caries and periodontal disease. Mouthwashes can be used as an adjunct to mechanical plaque control methods to decrease the load of oral microorganisms. Chlorhexidine (CHX) is a commonly used antimicrobial mouthwash with side effects such as changing the sense of taste, tooth discoloration, oral mucosal burning, allergy, and xerostomia. It also has adverse systemic effects, if swallowed. Purpose: This study aimed to assess the effect of photodynamic therapy (PDT) with curcumin and methylene blue (MB) photosensitizers and different laser parameters on S.A colony count. Materials and Method: In this in vitro experimental study, 99 samples of standard-strain S.A were subjected to PDT with curcumin and MB photosensitizers with/without irradiation of 660 and 445 nm laser with different exposure parameters, and CHX in 9 groups (n=11). The samples were cultured in microplates containing Mueller-Hinton agar, and the number of colony forming units (CFUs) was counted after 24 h of incubation at 37°C. Data were analyzed by the Kruskal-Wallis and Dunn tests. Results: The minimum colony count was noted in CHX group (CFUs=0) followed by MB and 660nm diode laser group irradiated for 100 s (CFUs=147.2727±169.35707). The difference in this respect was significant between MB+660nm diode laser for 100 s and other groups (p< 0.05) except for the MB + 660 nm diode laser for 60 s group. Conclusion: CHX is superior to laser for elimination of S.A. However, PDT with 660 nm diode laser + MB has considerable antimicrobial efficacy against S.A; increasing the duration of laser irradiation enhances the antimicrobial effect.


Introduction
Staphylococcus aureus (S.A) colonizes the skin, nasal cavity, and oral cavity, and is the most important human pathogen [1]. It has been isolated from the oral cavity, and is associated with bacterial infection of the salivary glands (particularly the parotid gland), angular cheilitis, denture stomatitis, and acute dentoalveolar infection. It is a part of the oral microflora [2][3] and can colonize in the oral cavity and subsequently cause dental caries and periodontal disease [4].
Mouthwashes are commonly used as an adjunct to mechanical plaque control methods to further decreasing the count of oral microorganisms [5]. Chlorhexidine (CHX) is a commonly used antibacterial mouthwash with a broad-spectrum antibacterial activity. However, it has drawbacks such as altering the sense of taste, discoloration of tooth and restoration surfaces, oral mucosal burning, allergy, xerostomia, and adverse systemic effects, if swallowed. These side effects limit the application of CHX [6]. Photodynamic therapy (PDT) requires three components of light, photosensitizer, and free radicals. In PDT, a certain wavelength of light is used to activate the photosensitizer and generate free radicals to eliminate the target cells. In dentistry, this modality is used to prevent the proliferation of microorganisms responsible for dental caries and periodontitis.
Some of benefits of PDT include being noninvasiveness, not requiring antibiotics, and the potential of destruction of bacteria in a short period of time [7].
Evidence shows that curcumin and MB can inhibit bacterial proliferation when irradiated with a specific wavelength of light [8].
Recently, PDT has gained increasing popularity for elimination of microorganisms [9]. Many of the bacteria and fungi that are part of the oral microflora have shown sensitivity to PDT [10][11][12]. Azizi et al. [13][14][15] [13][14][15]. Recently, blue lasers were introduced to the market with several applications as in oral soft tissue surgery [16]. Blue lasers can be used as the light source in PDT against S.M, Enterococcus faecalis, and C.A, with curcumin photosensitizer [16].
Considering the fact that PDT is easily available, low-cost, and non-invasive, and since the effect of PDT with blue laser and curcumin on S.A has not been previously evaluated, this in vitro study aimed to assess the effect of PDT with curcumin and MB on S.A

Statistical analysis
The SPSS version 18 was used for data analysis and level of significance was set at p<0.05. Since data were not normally distributed, statistical analysis was carried

Results
This experimental study evaluated the effect of PDT with 660nm diode laser and 445nm blue laser with MB and curcumin photosensitizers on S.A. Table 1    and this study appears to be the first on this topic.
Curcumin has various biological properties. It can inhibit the proliferation of cancer cells and serve as an anti-oxidant. In addition, it has antimicrobial activity against gram-positive and gram-negative bacteria [16].
Efficacy of a photosensitizer depends on three factors including its ability to bind to the bacterial membrane, to penetrate into the cells, and to generate free radicals around the bacteria when irradiated by light [16].
MB is an alkaline photosensitizer that can pass through the bacterial cell membrane, affect the bacterial genome, and eliminate the bacteria. In addition, when irradiated by laser, it generates free oxygen species that eliminate the bacteria [16][17][18].

S.
A is a gram-positive microorganism, with a thick cell wall, which is not highly permeable and inhibits the passage of hydrophobic materials [18]. This peptidoglycan layer has selective permeability against simple diffusion, and penetration of photosensitizer molecules depends on their size and degree of solubility [18].
CHX is a commonly used antimicrobial agent in dentistry, which effectively decreases the bacterial viability. CHX is the gold-standard against microbial biofilm. Our study confirmed the antibacterial activity of CHX against S.A, since it showed maximum antibacterial activity against S.A.

Conclusion
This study showed the superiority of CHX for elimination of S.A compared with laser irradiation. However, use of 660 nm diode laser + MB had significant antibacterial effect on S. aureus, and increasing the laser irradiation time enhanced its antimicrobial activity.

Conflict of Interest
The authors declare that they have no conflicts of in-